The present invention relates to the art of enzyme amplification, and, in particular, it concerns the use of the polymerase chain reaction for the enzymatic amplification of nucleic acids.
Several methods are known for the synthesis of nucleic acids from an existing sequence. These methods are capable of producing large amounts of a given nucleic acid of a specified sequence. U.S. Pat. No. 4,683,202 to Mullis and U.S. Pat. No. 4,800,159 to Mullis, et al., which are incorporated herein by reference, disclose the GENEAMP.TM. polymerase chain reaction method by which a length of target DNA (template) is replicated in an amplification process involving the use of Taq DNA polymerase as illustrated in FIG. 1. The DNA template is defined by two sites of a specific base sequence to which two oligonucleotide primer DNA molecules have been synthesized. Each primer is complementary to a base sequence at either end of the target DNA sequence and anneals to it such that extension by Taq DNA polymerase occurs in a direction toward the site of the other primer.
U.S. Pat. No. 4,800,159 to Mullis, et al. discloses the incorporation of restriction sites onto or into the 5' ends of primers used in an amplification process to produce amplified DNA with restriction sites at its ends. When cut with the appropriate enzymes, the amplified product can be inserted into plasmid or viral vectors and cloned. Mullis et al. further disclose the treatment of a nucleic acid with a restriction endonuclease prior to amplification. A cut sequence would not be amplified and the appearance of an amplified sequence would indicate that the amplified sequence does not have a restriction site specific for the restriction endonuclease. Mullis, et al. do not teach the incorporation of a restriction enzyme recognition site into a primer for the purpose of eliminating a contaminant PCR product from a subsequent PCR amplification process. Furthermore, Mullis, et al. do not disclose the treatment of contaminant PCR product with a restriction endonuclease to cleave the interprimer region such that the contaminant PCR product cannot be amplified in a subsequent PCR process.
It is possible to make many copies of the target DNA, by placing template DNA, Taq DNA polymerase, deoxyribonucleotide triphosphate precursors and other components into a reaction mixture and treating the reaction mixture with a sequence of heating steps. Typically, this involves an annealing phase at 45.degree.-55.degree. C., an extension phase at 70.degree.-75.degree. C., and a denaturation phase at 94.degree. C. During each cycle, (which includes each of the steps) the amount of target template DNA is increased theoretically by two-fold and over a million fold after twenty cycles.
The great sensitivity of the GENEAMP.TM. polymerase chain reaction method for the amplification of DNA requires that the amount of contaminant template DNA (DNA whose multiplication is not desired) be kept to a minimal level or be altered in such a way that it cannot undergo the multiplication process. The polymerase chain reaction method is so sensitive that only a small sample of template DNA is needed for multiplication to occur. Therefore, the presence of even a minute quantity of contaminant template DNA containing the target sequence could quite possibly result in the amplification of the contaminant template DNA and a mixture of amplified template and amplified contaminant template DNA. It is noted that the methods of the present invention are suitable for other methods of primer-dependent amplification of DNA and that the GENEAMP.TM. polymerase chain reaction method is used for exemplary purposes.
Two kinds of contamination with template DNA are most prevalent. The first type of contamination results from the presence of the product of a previous amplification in which the same target sequence was amplified with the same primers. Such DNA, can itself serve as template DNA. The second type of contamination results from the presence of template DNA which was not produced in a previous polymerase chain reaction process. This may occur for example, when a small amount of a DNA target-containing solution (e.g., from a genomic DNA extract) contaminates a solution which does not contain a target.
The first type of contamination is more difficult to eliminate. This is because polymerase chain reaction products are often present in very high copy number in completed amplification reactions (as high as 10.sup.12 copies). Since the polymerase chain reaction process has a potential of detecting and multiplying even a single molecule (or copy), the presence of even an exceedingly small amount of contaminant target DNA may result in significant contamination.
Therefore, even the use of such routine procedures as pipetting or the opening and closing of the reaction mixture container may result in the introduction of a sufficient amount of aerosolized material to interfere with the diagnostic use of the polymerase chain reaction method. Routine containment methods often fail to prevent the accumulation over time of such contaminant target DNA molecules in the local environment. Even the segregation of the amplified products in a separate laboratory has not generally succeeded in removing contaminant template DNA. This problem is particularly acute in those situations where the same set of primers are used repeatedly on the same target sequence, as is often the case in such amplifications. Efforts have been made to eliminate the contamination problem. These efforts have resulted in only limited success in reducing contamination.
It is therefore an object of the invention to significantly reduce and/or eliminate from amplification reactions the presence of amplifiable contaminant target DNA arising from previous similar amplification reactions (i.e., reactions using the similar primers and similar target sequences).
It is a further object of the present invention to enzymatically act upon contaminant DNA or contaminant target DNA arising from previous similar amplification reactions (i.e., reactions using the same primers and the same target sequence) or to otherwise alter or eliminate such contaminant DNA such that the contaminant DNA is no longer capable of being amplified.
It is yet another object of the present invention to heat-inactivate enzymes used to inactivate contaminant DNA in such a manner that the reaction mixture need not be exposed to the atmosphere and possible recontamination until completion of the amplification process.